The catalytic domains of diphtheria toxin, anthrax toxins (protective antigen [PA]. lethal factor [LF] and edema factor [EF]) and all serotypes of botulinum neurotoxin follow a similar route of entry into the cytosol of their respective targeted eukaryotic cells. Following binding of each toxin to their specific cell surface receptor, the toxin is internalized into the cell by receptor-mediated endocytosis in clathrin coated pits. Following acidification of an early endosomal compartment, the translocation of their respective catalytic domains to the cytosol is facilitated by at least a functional transmembrane domain. Using an in vitro translocation assay, we have recently demonstrated that a cellular translocation factor (CTF) complex is required for the delivery of the diphtheria toxin C-domain across the membrane of early endosomal vesicles. The X-ray structure of diphtheria, anthrax toxin, and botulinum toxin A is known. BLAST analysis of these toxins has suggested the presence of 12 amino acid "entry motif" in each toxin. The present application proposes the continuing study of diphtheria toxin C-domain, alanine scanning mutagenesis of the putative "entry motif", and the development of an in vitro translocation assay for anthrax lethal factor. In the case of diphtheria toxin, anthrax toxins, and botulinum toxins a great deal is known of their respective structure function domains and their mode of action; however, a detailed understanding of the molecular mechanism(s) required for translocation of their respective catalytic domains from the lumen of acidified early endosomes to the cytosol have remained elusive. Only a few studies have focused directly on the molecular mechanism(s) of translocation of the toxin catalytic domain entry have been published. In the case of diphtheria toxin, the results of these studies have led to two divergent hypothetical mechanisms of catalytic domain translocation. The first is based on the hypothesis that the toxin itself carries sufficient structural information to mediate translocation of its catalytic domain across the endocytic vesicle membrane. In contrast, the second hypothesis is based on findings that both structural domains of the toxin in combination with a cytosolic translocation factor (CTF) complex are necessary for productive translocation. We have used purified early endosomes charged with the fusion protein toxin, DAB3891L-2, to critically examine both hypotheses.